Members of my lab have been studying the combined effect of activated ethanol-inducible cytochrome P450-2E1 (CYP2E1), a pro-oxidant enzyme, and suppressed mitochondrial aldehyde dehydrogenase (ALDH2), an antioxidant enzyme for the removal of toxic acetaldehyde and lipid peroxides, on promoting tissue injury by alcohol and other potentially toxic substances. Alcohol-induced oxidative and nitrative (nitroxidative) stress inactivated the ALDH2 activity, resulting in accumulation of potentially toxic acetaldehyde and lipid peroxides. In addition, CYP2E1-mediated nitroxidative stress can stimulate different types of post-translational modification (PTM) of cellular proteins, contributing to mitochondrial dysfunction, endoplasmic reticulum (ER) stress and tissue/organ damage. These PTMs include oxidation, S-nitrosylation, nitration, phosphorylation, adduct formation, etc. that usually occur after exposure to alcohol and nonalcoholic substances or under pathological conditions. In the past, we showed a causal role of CYP2E1 in stimulating various PTMs and tissue injury by evaluating the time-dependent events of PTMs and actual cellular damage in the presence or absence of an antioxidant N-acetylcysteine (NAC) or a specific CYP2E1 inhibitor chlormethiazole (CMZ). However, PTMs and functional alterations of covalently-modified proteins are observed in the liver and other extra-hepatic tissues such as gut and brain. Therefore, we have also characterized the nitrated and/or p-JNK-target proteins in gut and brain of wild-type (WT) mice, WT rats and HIV-1 transgenic (Tg) rats where elevated levels of nitroxidative stress and p-JNK are observed after exposure to binge alcohol, fructose, or a western-style fast food-high fat diet (FF-HFD) containing high cholesterol. We recently reported the critical role of CYP2E1 in binge alcohol-mediated leaky gut, endotoxemia, and inflammatory liver injury in WT mice and rats but not in Cyp2e1-null mice. We have continuously studied the mechanism of intestinal barrier dysfunction by investigating the role of different PTMs of the junctional complex proteins including tight junction (TJ), adherent junction (AJ), and desmosome in alcohol-induced gut leakiness and endotoxemia. Binge alcohol exposure significantly increased the levels of intestinal CYP2E1, iNOS, nitrated proteins, apoptosis-related marker proteins, serum endotoxin and fecal albumin contents, suggesting elevated apoptosis of enterocytes, gut leakiness and endotoxemia. Differential mass-spectral analyses of the TJ-enriched fractions of gut epithelial cells showed that several TJ, AJ and desmosome proteins were decreased in alcohol-exposed rats compared to controls. Immunoblots revealed that the levels of TJ proteins (claudin-1, occludin and ZO-1), AJ proteins (-catenin and E-cadherin) and desmosome plakoglobin were markedly decreased in binge-alcohol exposed rats, WT mice, and ileums from autopsied people who died due to heavy alcohol intoxication, but not in the corresponding Cyp2e1-null mice. Our results demonstrate for the first time the critical roles of increased apoptosis of enterocytes and nitration followed by ubiquitin-dependent degradation of the junctional complex proteins in promoting gut leakiness and endotoxemia. Based on our result with binge alcohol-mediated leaky gut and inflammatory tissue injury, we have also studied the mechanisms of leaky gut, endotoxemia and advanced liver disease (fibrosis) by nonalcoholic substances such as FF-HFD or fructose. In fact, our mechanistic studies with FF-HFD- or fructose-exposed mice and rats showed similar mechanisms of gut leakiness, endotoxemia and liver fibrosis through increased apoptosis of gut enterocytes and nitration of the junctional complex proteins in rats and WT mice. However, the levels of CYP2E1 were not increased in the latter models, although Cyp2e1-null mice were resistant to leaky gut and inflammatory liver injury. These results suggest an indirect, permissive role of CYP2E1 in gut leakiness, endotoxemia and inflammatory tissue injury caused by the two nonalcoholic substances. From 2016, we have started a new project to characterize extracellular vesicles (EVs) in circulating blood secreted from the damaged liver in mice and rats after exposure to binge alcohol (ethanol) or acetaminophen (APAP). The amounts of total and liver-specific proteins in circulating EVs from mice exposed to APAP or binge alcohol were significantly increased in a dose- and time-dependent manner. Proteomic analysis of EVs revealed that the amounts of liver-specific proteins were increased in APAP-exposed mice compared to those of controls. Consistently, binge ethanol exposure markedly elevated liver-specific proteins in circulating EVs from WT mice and patients with alcoholic hepatitis, compared to their respective controls. The number of EVs and the amounts of EV CYP2E1 and other P450 isoforms were markedly elevated in both alcoholics and alcohol-exposed rats and WT mice, but not in the corresponding Cyp2e1-null mice. The amounts of EV CYP2E1 depended on increased oxidative and ER stress, since their levels were decreased by co-treatment with NAC or CMZ, but increased by an ER stress inducer thapsigargin. Moreover, cell death rates were elevated when recipient primary hepatocytes were exposed to exogenous EVs from alcohol-exposed rodents and human alcoholics, showing that exogenous EVs from alcohol- or APAP-exposed mammals are functional in cell-cell communication and can promote death of recipient hepatocytes by activating the apoptosis signaling pathway such as p-JNK, Bax and caspase-3. These results show an important role of CYP2E1 in elevating EV CYP2E1 through increased oxidative and ER stress. In addition, elevated EV-CYP2E1 can be used as a potential biomarker for drug- and alcohol-mediated liver injury. We further investigated whether exogenous exosomes from donor mice with APAP-induced liver injury can damage the recipient hepatic cells or promote hepatotoxicity in other mice. Our results showed that exogenous exosomes derived from APAP-exposed mice were internalized into the primary mouse hepatocytes or HepG2 hepatoma cells and significantly decreased the viability of these recipient cells. They also elevated mRNA transcripts and proteins associated with the cell death signaling pathways in primary hepatocytes or HepG2 cells via exosomes-to-cell communications. Furthermore, exosomes isolated from patients with mastocytosis stimulated cultured hepatic stellate cells and recipient mice with increased levels of profibrogenic proteins. These results strongly demonstrate that exogenous exosomes can regulate the cellular functions of the recipient cells/animals and thus can be used as a valuable target of therapeutic interventions. Based on our basic mechanistic studies, we have conducted translational research by evaluating the beneficial effects of dietary supplements, including indole-3-carbinol (I3C) or pomegranate (POM), against AFLD in rodents and T84 colon cells. Daily supplementation with I3C (40 mg/kg/day orally) or POM (600 mg/kg/day orally) significantly prevented binge alcohol-mediated gut leakiness, endotoxemia and AFLD by preventing PTMs of intestinal TJ/AJ proteins. I3C and POM also decreased the nitration and ubiquitin conjugation followed by proteasomal degradation of gut TJ/AJ proteins and prevented elevated levels of activated p-JNK, p-p38K and apoptosis of enterocytes and hepatocytes. The protective mechanisms of I3C and POM against binge alcohol-mediated leaky gut and inflammatory liver injury are very similar and appear to be mediated by suppressing oxidative and nitrative stress in the intestine and liver. In addition, we are evaluating the effects of physiologically relevant doses of ellagic acid, a component of POM, on binge alcohol-mediated leaky gut and liver inflammation.